Nitrite Assimilation and Amino Nitrogen Synthesis in Isolated Spinach Chloroplasts

Abstract

The assimilation of nitrite leading to de novo synthesis of amino nitrogen in a chloroplast-enriched fraction isolated from freshly harvested young spinach (Spinacia oleracea L.) leaves was demonstrated. The preparations showed approximately 55% intact chloroplasts as determined by light scattering properties and fixed C02 at rates of approximately 100 Amoles hr-mg chlorophyll1.The chloroplast-enriched fraction contained the enzymes, nitrite reductase and NADPH-glutamate dehydrogenase, needed for the reduction of nitrite and incorporation of ammonia into glutamate. Kinetic studies showed that the reduction of nitrite by the chloroplast-enriched fraction is light-dependent, and the process proceeds at rates of 6 to 12 Amoles hr-1 mg chlorophyll1. The addition of nitrite to the chloroplast preparation caused a 3-fold increase in the production of a-amino nitrogen when compared with the control without nitrite. There was a stoichiometric relation between amino-nitrogen synthesis and nitrite disappearance from the medium. The ratio of aminonitrogen :NO,-ranged from 0.6 to 0.9. The initial rate of amino-nitrogen production was faster when a-ketoglutarate was added to the nitrite reducing chloroplast medium than when it was omitted. However, these high rates were not sustained and the total amino-nitrogen production at the end of a 30-minute period was only slightly higher. These data show that chloroplasts are functionally able and contain the enzyme complement necessary to utilize light energy for the reduction of nitrite to amino nitrogen. Thus, chloroplasts should be considered as a major site for in vivo amino-nitrogen synthesis in green plants.

Light-dependent Assimilation of Nitrite by Isolated Pea Chloroplasts

“…The rates of light-dependent nitrite-reduction catalyzed by intact pea chloroplasts for condition I approximate those found for spinach (9,10,14) and maize (15), but the rate of nitrite-dependent 02 evolution (17.5 ,umol mg of Chl' hr-') was somewhat greater (10) presumably due to differences in species, chloroplast quality, and the use of glyceraldehyde. Method 1 pea chloroplasts, incubated under condition 1, appear to differ from spinach in that most of the nitrite-N consumed is recovered in ammonia.…”

“…Although the rates relative to those for method 1 chloroplasts are low, the results are consistent with the requirement for PPi for the incorporation of ammonia into glutamine catalyzed by method I chloroplasts (1) as used in condition 2. Perhaps it is significant that experiments in which a-ketoglutarate has been shown to affect nitrite reduction (10) and incorporation of nitrite-N into amino-N (9) have been conducted in the presence of PPi buffer (10) or 0.5 mM Pi (9).…”

“…Thus, glutamate synthetase activity can be monitored by (glutamine plus a-ketoglutarate)-dependent 02 evolution (2). In the presence of the cofactors ADP, PPi, and Mg2+, pea chloroplasts also catalyze (ammonia plus a-ketoglutarate)-dependent 02 evolution which we attributed to assimilation of ammonia into glutamate via photosynthetically coupled4 glutamine synthetase and glutamate synthetase (1).Spinach chloroplasts in the light actively reduce nitrite (9,10,14); approximately 60 to 90% of the nitrite-N consumed is incorporated into amino-N (9, 10). Spinach chloroplasts also catalyze nitrite-depentent 02 evolution (10).…”

“…Spinach chloroplasts in the light actively reduce nitrite (9,10,14); approximately 60 to 90% of the nitrite-N consumed is incorporated into amino-N (9, 10). Spinach chloroplasts also catalyze nitrite-depentent 02 evolution (10).…”

Nitrite Transport in Chloroplast Inner Envelope Vesicles (I. Direct Measurement of Proton-Linked Transport)

“…Within the chloroplast stroma nitrite reductase reduces NO,-to NH4', which is further incorporated into amino acids (Anderson and Done, 1978). Nitrite reductase activity has been measured in isolated chloroplasts from spinach and pea preparations and shown to range from 1 to 12 pmol h-' mg-I Chl at 0.33 to 0.5 mM NO,-(Magalhaes et al, 112, 19961974Anderson and Done, 1978;Brunswick and Cresswell, 1988a). Therefore, the rate of proton-linked NO,-transport is approximately 5 to 80 times greater than the measured activity of NO,-reductase in chloroplasts.…”